Subject(s)
Vaccines, DNA/administration & dosage , Animals , Cations , Cetrimonium , Cetrimonium Compounds/chemistry , DNA/chemistry , Enzyme-Linked Immunosorbent Assay , Female , Gene Products, gag/immunology , HIV Envelope Protein gp120/immunology , Immunoglobulin G/biosynthesis , Lactic Acid/chemistry , Mice , Mice, Inbred BALB C , Microspheres , Particle Size , Plasmids/immunology , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer , Polymers/chemistry , Protein Precursors/immunology , Vaccines, DNA/immunologyABSTRACT
We have developed protective interactive noncondensing (PINC) polymers, such as poly(N-vinyl pyrrolidone) (PVP) and poly(vinyl alcohol) (PVA), to protect plasmids from extracellular nuclease degradation while allowing the flexible complex to diffuse throughout the muscle tissue. Molecular modeling, zeta potential modulation, and ethidium bromide intercalation studies were performed to assess the mechanism of interaction between PVP and plasmid. The effect of salt concentration, pH, and polymer-plasmid ratios were investigated. We have correlated these variables with beta-galactosidase (beta-gal) expression after intramuscular administration to rats. PVP can form hydrogen bonds with the base pairs within the major groove of DNA at pH 4.0. The PVP-plasmid interaction results in a complex that is more hydrophobic (less negatively charged) than the uncomplexed plasmid due to the vinyl backbone of PVP. Up to a ten-fold enhancement in gene expression in rat muscle over the use of 'naked' DNA has been demonstrated using these systems. A linear structure-activity relationship (SAR) was found between the percent vinyl pyrrolidone monomer content in poly (vinyl pyrrolidone-covinyl acetate) polymers and beta-gal expression in muscle. Modulation of the interaction between PINC polymers and plasmid directly impacts the levels of gene expression in vivo. The linear SAR is being used to design novel PINC polymers with enhanced binding affinity to plasmids.
Subject(s)
Muscle, Skeletal/metabolism , Plasmids , Polyvinyl Alcohol/pharmacology , Povidone/pharmacology , Animals , Ethidium , Gene Expression , Hydrogen-Ion Concentration , Immunohistochemistry , Male , Models, Molecular , Rats , Rats, Inbred F344 , beta-Galactosidase/metabolismSubject(s)
2,2'-Dipyridyl/analogs & derivatives , DNA-Binding Proteins/metabolism , DNA/metabolism , Intercalating Agents/pharmacology , Organometallic Compounds/pharmacology , Pharmaceutical Preparations/metabolism , 2,2'-Dipyridyl/chemistry , 2,2'-Dipyridyl/pharmacology , Base Sequence , Binding Sites , DNA/drug effects , DNA/radiation effects , DNA Adducts , DNA Damage , Distamycins/metabolism , Electrophoresis, Polyacrylamide Gel , Intercalating Agents/chemistry , Molecular Sequence Data , Organometallic Compounds/chemistry , Photochemistry , Protein Binding , Ultraviolet RaysABSTRACT
Laser-induced Eu(3+) luminescence spectroscopy is used to probe the interaction of Eu(3+) ion with guanine-containing nucleotides and single-stranded oligomers. By using time-resolved and non-time-resolved Eu(3+) luminescence techniques, two classes of Eu(3+) binding site are observed in oligo(dG)10, oligo(dG)8, oligo(dG)6, oligo(dG)4, and d-GMP. One class of site binds Eu(3+) ions more strongly than the other. Since the "tight" class of bound Eu(3+) ions have two coordinated water molecules, it is inferred that six or seven atoms from the oligomers are coordinating the Eu(3+). The "weaker" class of Eu(3+) ion sites involve the coordination of six or seven water molecules and therefore, are coordinated by one or two atoms from the oligomer. The tight class of Eu(3+) binding site is attributed to an interstrand association of Eu(3+) with the oligomers forming dimeric or polymeric structures. The dissociation constants (Kd) for the 1:1 complexes Eu(d-GMP)+ and Eu(d-GTP)- have been determined as well as the Kd for the dimerization reaction of Eu(d-GMP)+. The Tb(3+) luminescence enhancement properties of these molecules are also examined in relation to their EU(3+) binding characteristics.
Subject(s)
DNA/chemistry , Deoxyguanine Nucleotides/chemistry , Europium/chemistry , Oligodeoxyribonucleotides/chemistry , Terbium/chemistry , Binding Sites , Luminescent Measurements , Spectrum Analysis , Water/chemistryABSTRACT
Oligo(dC)8, oligo(dA)8, and oligo(dT)8 as well as d-CMP, d-AMP, and d-TMP, when complexed to Eu(3+), possess two classes of Eu(3+) binding environment. The binding environments consist of two classes, tight sites which coordinate two H2O molecules, and weaker sites which coordinate six or seven, analogous to the previously studied guanine-containing molecules. It is inferred that the tight class of Eu(3+) ion site observed with these oligomers and nucleotides corresponds to dimeric or polymeric structures. Comparison of the results for the guanine and non-guanine containing oligomers suggests that Eu(3+) possibly coordinates base nitrogen atoms in the former and in an outer sphere mode (hydrogen bonding via the H2O molecules coordinated to Eu(3+)) in the species examined here.
